AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. In clinical trial, rAAV vectors delivering RPE65 gene have been demonstrated to cure Leber's congenital amaurosis (LCA) diseases and patients are able to regain normal vision after receiving the vectors. However, its use in other genetic diseases such as hemophilia still face considerable challenge. Systemic delivery of rAAV into human subjects would require a lot more vectors than targeting the retina tissue. Prelimary studies have demonstrated more than 99.9% of rAAV vectors were wasted in the varous stages of rAAV transduction. In addition, rAAV genomes packaging preference remains an unsettling issues. In order to further develop recombinant AAV for human gene therapy, we hypothesize that it would eliminate unwanted replication competent AAV particle formation and improve rAAV production by sequetering the helper function and vector sequencing into different cellular compartment. Advanced genomics and proteomics will be introduced to study rAAV packaging preference and encapsidation mechanism. Finally, we identified that cytoplasm is major site for rAAV genome loss. An innovative assay will be established to track rAAV genomes in cytoplasm host and mechanism will be studied to reduce rAAV genomes in cytoplasm of the host cells. Hence, our three specific aims are 1. To develop and optimize the next generation of rAAV packaging system~ 2. To characterize rAAV genome integrity and rAAV packaging mechanisms~ 3. To study the molecular status of recombinant AAV genomes in the cytoplasm. Completion of specific aims in this application will markedly enhance the way of rAAV vectors to be used in the human gene therapy field.

Public Health Relevance

The completion of this project may provide a solution for producing high quality rAAV vector economically. Our work may help design optimized vector for human gene therapy and improve the quality of life of patients with genetic diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080789-07
Application #
8336917
Study Section
Special Emphasis Panel (ZRG1-BST-N (90))
Program Officer
Skarlatos, Sonia
Project Start
2006-02-15
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
7
Fiscal Year
2012
Total Cost
$344,295
Indirect Cost
$119,266
Name
Temple University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Wang, Q; Dong, B; Firrman, J et al. (2016) Evaluation of the biological differences of canine and human factor VIII in gene delivery: implications in human hemophilia treatment. Gene Ther 23:597-605
Moore, Andrea R; Dong, Biao; Chen, Lingxia et al. (2015) Vaccinia virus as a subhelper for AAV replication and packaging. Mol Ther Methods Clin Dev 2:15044
Dong, Biao; Duan, Xunbao; Chow, Hoi Yee et al. (2014) Proteomics analysis of co-purifying cellular proteins associated with rAAV vectors. PLoS One 9:e86453
Dong, Biao; Moore, Andrea R; Dai, Jihong et al. (2013) A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy. Nucleic Acids Res 41:6609-17
St Laurent, Georges; Shtokalo, Dmitry; Dong, Biao et al. (2013) VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer. Genome Biol 14:R73
Kapranov, Philipp; Chen, Lingxia; Dederich, Debra et al. (2012) Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing. Hum Gene Ther 23:46-55
Roberts, Sean A; Dong, Biao; Firrman, Jenni A et al. (2011) Engineering Factor Viii for Hemophilia Gene Therapy. J Genet Syndr Gene Ther 1:
Lu, Hui; Qu, Guang; Yang, Xiao et al. (2011) Systemic elimination of de novo capsid protein synthesis from replication-competent AAV contamination in the liver. Hum Gene Ther 22:625-32
Dong, Biao; Nakai, Hiroyuki; Xiao, Weidong (2010) Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther 18:87-92
Chen, Lingxia; Lu, Hui; Wang, Jinhui et al. (2009) Enhanced factor VIII heavy chain for gene therapy of hemophilia A. Mol Ther 17:417-24

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